High-alkaline protease and its use arginine-substituted subtilisin composition and use

ABSTRACT

The invention relates to a novel high-alkaline protease, its use in the industrial or domestic fields and also compositions for said uses containing this protease. The protease of the invention is a selected triple mutant derived from certain precursor proteases from the subtilisin group, especially of the “subtilisin 309” type.

This application is the U.S. national stage application of PCT/EP95/01141, filed Mar. 27, 1995, which claims priority to German patentapplication number P44 11 223.8, filed Mar. 31, 1994.

The subject matter of this invention concerns a new high-alkalineprotease, its use in the industrial and domestic field, and compositionsfor the applications mentioned which contain this protease.

The use of protease-containing compositions in industrial applicationsor processes is well known. For example, in commercial laundryestablishments, proteases have long been used, for example, to cleanblood-stained hospital linen as well as protective clothes worn inmeat-processing plants. For the production of leather, it is stillnormal practice in the leather treatment industry, for example, toremove the hair from skins and hides in an alkaline processing stage inthe so-called beamhouse, which creates the prerequisites for hairremoval and which causes the necessary skin digestion, often still usingquestionable and unsafe chemicals (e.g., inorganic sulfides). Althoughrecently enzymatically supported liming processes (hair removalprocesses) have been proposed—especially using tryptic enzymes or fungalor bacterial proteases and sometimes even carbohydrolases—in practice,these enzymatic processes for the removal of hair from hides and skinshave been applied almost exclusively to hides and skins of small animalsand even in this area, the use has been rather restricted. For theremoval of hair of large animals, on the other hand, the enzymatic hairremoval process has so far not gained any acceptance at all, mainlybecause in many cases, hair removal is incomplete and because thecollagen grain membrane is damaged or because too much of the skinsubstance is destroyed. In addition, the use of a certain percentage ofalkaline proteases during liming with a reduced quantity of chemicals(e.g., sulfides) has been investigated. Although the use of enzymesmakes it possible to markedly reduce the quantity of chemicals (e.g.,sulfide) and although excellent surface yields with little cicatricialcontraction are obtained, leather produced in this manner tends towardgrain pipeyness, toward a loose flame scarring structure, and toward acoarse appearance which sometimes resembles the grain of nubuck leather.To the extent that proteases are presently used in the leathermanufacturing industry (main soaking cycle and liming), these prior-artproteases, while having high pH values (pH=11 to 13), are notsufficiently effective and have a relatively low activity at thetreatment temperatures (28° C. to 30° C.) normally encountered in thelimeyard.

Due to the fact that the conditions in industrial processes are moredrastic than those in domestic applications (e.g., as a householddetergent), the proteases used must meet especially stringentrequirements with respect to stability, acceptance of the prevailingenvironment, and performance. In addition to a satisfactory stabilityand activity at high alkaline pH values, the proteases should, on theone hand, have an excellent temperature resistance so as to yield goodresults at a low concentration over the longest possible time at atemperature that for a given industrial application can be very highand, on the other hand, they should be sufficiently active in certainapplications (e.g., leather manufacture) even at relatively lowtemperatures (approximately 30° C.). Furthermore, the alkaline proteasesused should be as resistant as possible to the chemicals and ingredientsthat are conventionally used in industrial processes (e.g., surfactants,bleaching agents, or disinfecting agents, chemicals, and otherconstituents).

Thus, the need for other alkaline proteases that are suitable forindustrial applications, e.g., proteases for industrial textilelaundering processes, industrial surface cleaning, or leather treatmentsand leather manufacture, is undiminished.

Therefore, the problem to be solved by this invention was to create anew alkaline protease which is suitable especially for use in industrialprocesses and which, in addition, can also be used to advantage fordomestic applications.

It was discovered that the alkaline bacillus protease described belowcan be used highly effectively in a number of industrial processes.Thus, one of the subject matters of this invention concerns an alkalineprotease and its use especially in compositions for industrialapplications as well as in the domestic field, such as proposed in theclaims and described in greater detail below.

Therefore, the subject matter of this invention concerns a high-alkalineprotease which is characterized by the fact that it contains anunderlying amino acid sequence with a minimum of a 95%, preferably witha minimum of a 98%, homology with respect to the amino acid sequenceshown in FIG. 1 and that it is distinguished from this sequence by atriple mutation in the positions 42/114/115 of FIG. 1 or that isdistinguished in the three positions homologous thereto by the fact thatarginine has been substituted for the amino acids in the relevantpositions.

The alkaline bacillus protease mentioned has a molecular weight ofapproximately 26,000 to 28,000 g/mol, measured by means of SDSpolyacrylamide gel electrophoresis against references proteins with aknown molecular weight. The optimum pH value which was determined withsoluble model substrates in an analytical test is approximately pH 10.5,with the optimum pH value being defined as that pH range in which theprotease has a maximum proteolytic activity. The pH activity is higherthan in the original protease (according to FIG. 1); the optimum effectextends further into the more alkaline range and is pH 10.5 to 11.5. Inaddition, the mentioned alkaline bacillus protease according to thisinvention has an excellent pH stability and temperature resistance.Thus, this protease is an extremely high-alkaline protease which iseffective in a pH range so far not reached by prior-art proteases.

In this context, homology with respect to the amino acid sequence shownin FIG. 1 is defined as the structural relationship between the relevantamino acid sequences and the amino acid sequence shown in FIG. 1. Todetermine the homology, the segments of the amino acid sequence of FIG.1 which structurally correspond to one another and of the amino acidsequence with which they are to be compared are made to coincide in sucha way that a maximum structural agreement between the amino acidsequences exists, and differences caused by the deletion or insertion ofindividual amino acids are taken into consideration and are compensatedfor by appropriate rearrangements of sequence segments. The number ofamino acids which now match one another in the sequences (“homologouspositions”), relative to the total number of the amino acids that arecontained in the sequence of FIG. 1, is the homology in %. Differencesin the sequences can be caused by variation and insertion and bydeletion of amino acids. It is therefore obvious that, if alkalineproteases are used which are at least 95% homologous with respect toFIG. 1, the amino acid positions named with respect to FIG. 1 refer tothe positions of the protease used which are homologous thereto.Deletions or insertions in the amino acid sequences of the proteasesthat are homologous with respect to FIG. 1 can lead to a relativerearrangement of the amino acid positions so that the numericalnotations of the amino acid positions that correspond to one anotherneed not be identical in homologous fragments of amino acid sequencesthat are homologous with respect to one another, i.e., it is possiblefor slight numerical rearrangements to develop relative to theindividual numbering of the amino acid positions.

In a preferred modification of this invention, the high-alkalineprotease is characterized by an underlying amino acid sequence which issubstantially identical to the amino acid sequence shown in FIG. 1 andwhich differs from this amino acid sequence by a triple mutation inpositions 42/114/115 of FIG. 1 in that the amino acids in the relevantpositions have been replaced by arginine. The term “substantiallyidentical” amino acid sequence is here defined to indicate that, withthe exception of the mentioned triple mutation N42R/N114R/N115R, only afew other, i.e., especially only up to 6 (corresponding to anapproximately 98% homology or more), amino acids can differ from theamino acids shown in the sequence of FIG. 1. The amino acid sequenceunderlying the high-alkaline protease according to this invention thuscorresponds substantially to those proteases which can be designated asbeing of the “subtilisin 309”-type since the amino acid sequence of the“subtilisin 309” known from prior art is identical to the amino acidsequence shown in FIG. 1. A protease which is nearly identical theretoand which, with the exception of position 85, coincides with the aminoacid sequence in FIG. 1, has been described in prior art as a proteasefrom Bacillus nov. species PB92 (see European Patent Application EP283,075). The protease from Bacillus PB92 differs only insignificantlyfrom the amino acid sequence shown in FIG. 1 in that in position 85, thenatural difference asparagine instead of serine is present, and it istherefore considered to be “subtilisin 309”-type protease. Anotherprotease which is substantially identical to “subtilisin 309” and which,with the exception of the five positions 97, 99, 101, 102, and 157,coincides with the amino acid sequence in FIG. 1, has been described inprior art as protease from Bacillus lentus (see International PatentPublication WO 91/02792 and U.S. Pat. No. 5,352,604) (“BLAP”). Protease“BLAP” from Bacillus lentus differs only insignificantly from the aminoacid sequence shown in FIG. 1 in that natural differences exist in fivepositions: 97D, 99R, 101A, 102I, and 157S. A variant of “BLAP” differsadditionally due to a sixth natural difference threonine instead ofserine in position 3 (alkaline protease which has also been described inInternational Patent Publication WO 91/02792 and U.S. Pat. No.5,352,604). D=Asp=asparagic acid, R=Arg=arginine, A=Ala=alanine,I=Ile=isoleucine, S=Ser=serine, T=Thr=threonine. “BLAP” and its variantwith the mutation S3T thus have an approximately 98% homology withrespect to the amino acid sequence of FIG. 1 and are accordinglyconsidered to be “subtilisin 309”-type proteases. Therefore, thisinvention preferably concerns high-alkaline proteases with the triplemutation N42R / N114R / N115R, the underlying amino acid sequence ofwhich is identical to the amino acid sequence shown in FIG. 1 or differstherefrom only in position 85 due to the natural difference asparagineinstead of serine or only in the following five position due to thenatural differences 97D/99R/101A/102I/157S or only in the six positionsbecause of the natural differences 3T/97D/99R/101A/102I/157S.

The alkaline bacillus protease underlying the proteases according tothis invention can be obtained from the bacillus strain (with the aminoacid sequence of FIG. 1) that was deposited under No. 5466 with the DSM[Deutsche Sammlung von Mikroorganismen und Zellkulturen, GermanCollection of Microorganisms and Cell Cultures, the German equivalent ofthe ATCC] on Jul. 28, 1989; further details, especially the isolation,concerning this strain have been described in the European PatentApplication EP 415,296. The variant, which differs in position 85, ofthe protease underlying the protease mutants according to this inventioncan be similarly obtained by cultivating the Bacillus nov. species PB92(as described in the European Patent Application EP 283,075 and in thecorresponding U.S. Pat. No. 5,217,878). The variants, which differ inpositions 97/99/101/102/157 or in positions 3/97/99/101/102/157, of theprotease underlying the protease mutants according to this invention canbe similarly obtained by cultivating Bacillus lentus or, afterappropriate transformation, even in Bacillus licheniformis (as describedin the International Patent Publication WO 91/02792 and in thecorresponding U.S. Pat. No. 5,352,604). The variants in position 85 andthe variants “BLAP” can be produced by means of producing correspondingpoint mutations in the positions mentioned from the protease with theamino acid sequence shown in FIG. 1. From the above-mentioned proteasesof the “subtilisin 309”-type underlying the protease mutants accordingto this invention, it is possible to produce the high-alkaline proteasesaccording to this invention with the triple mutation N42R/N114R/N115Raccording to substantially known methods by means of combined orconsecutive point mutations in the amino acid sequence of the precursorprotease of the “subtilisin 309”-type. The European Patent ApplicationEP 415,296 describes how such point mutations processes relative toindividual amino acid positions are carried out, and the proceduredescribed in this application can be used to produce the triplemutations for the production of the proteases according to thisinvention. With reference to the patent applications mentioned above,the contents of the prior patent applications relating to this subject,especially of the European Patent Application EP 415,296 (and thecorresponding U.S. Pat. No. 5,352,603), is therefore expresslyincorporated into this application.

In a highly preferred modification of this invention, the high-alkalineprotease according to this invention with the triple mutationN42R/N114R/N115R is derived from an underlying alkaline bacillusprotease from strain DSM 5466 with an underlying amino acid sequencethat is identical to that shown in FIG. 1. In the mutations which aregiven in the shorthand notational convention, the numbers refer to theposition in the amino acid sequence (see FIG. 1). The original aminoacid precedes the number and the new amino acid which was introduced bymutation into the amino acid sequence in the relevant position followsthe number. To identify the amino acids, the one-letter code is used: Nstands for asparagine (Asp) and R stands for arginine (Arg). As alreadymentioned above, the amino acid exchanges can be obtained according tosubstantially known methods by point mutation in the amino acidsequence, which procedure has been described, for example, in theEuropean Patent Application EP 415,296.

The high-alkaline bacillus protease mutants N42R/N114R/N115R accordingto this invention are exceptionally active under conditions whichgenerally prevail in industrial applications—high pH values, hightemperatures, short application times. The protease mutants according tothis invention are also surprisingly resistant to the constituentscontained in the formulation that are conventionally used in industrialprocesses, e.g., in industrial textile laundering processes. Thehigh-alkaline bacillus protease mutants according to this invention cantherefore be used to considerable advantage in industrial processes,such as in industrial textile laundering processes, in any type ofindustrial surface cleaning process, in leather treatment processes,especially, for example, in the manufacture of leather. An industrialapplication of the protease according to this invention in industrialtextile laundering establishments has been described in greater detail,for example, in the German Patent Application DE 4,411,223; thus, thehigh-alkaline bacillus protease mutant according to this invention canbe used, for example, in industrial large drum washing machines or infully continuous or countercurrent washing plants. It is especiallypreferred if in the so-called multiliquor processes, e.g., in thetwo-bath process consisting of a preliminary washing and a clear rinsingcycle, the alkaline bacillus protease according to this invention isadded to the preliminary washing cycle. Using substantially knownmethods, preliminary washing can be carried out under the conditionsgenerally used in industrial textile laundering processes, e.g., attemperatures from 30° C. to 70° C., with detergent ingredients that arenormally used in this washing cycle. In the case of contaminations witha high protein content, for example, blood-stained laundry fromhospitals or, e.g., laundry from institutional kitchens ormeat-processing plants, the proteases mentioned can also be successfullyused, for example, in a rinsing cycle, which precedes the preliminarywashing cycle, with clear cold or recycled warm water and otherwiseconventionally added detergent ingredients. In addition, the alkalinebacillus protease mutants according to this invention can also be usedto advantage in other industrial textile laundering processes, e.g., inindustrial laundering processes which specifically target special typesof textiles and contaminations, for example, to disinfect fabrics thathave been used in hospitals. Details can be found in the German PatentApplication DE 4,411,223.

Another application area in which this invention can be successfullyused is the cleaning of (hard) surfaces in industrial plants, preferablyin slaughtering plants of the food industry, in institutional kitchensin grill restaurants, etc.

All surfaces which during the production or processing of food come intocontact with this food have to be cleaned at regular intervals.Different branches (e.g., the beverage, canning, and sugar industry andthe milk-, meat-, and fat-processing industry) have to deal withdifferent types of contaminations. To remove these contaminations, priorart provides for a relatively large range of different cleaning agents.First, these cleaning agents have to remove protein, fat, andcarbohydrate stains. In addition to organic contaminations, there areinorganic contaminations that must be removed. Therefore, the cleaningagents consist of a mixture of different ingredients which servespecific functions during the cleaning process. These cleaning agentsare commercially available in the form of powders or liquids, in rarercases also in the form of pastes, and, with a few exceptions, have to bediluted by the consumer with water to concentrations of 0.5 to 2 wt %.The enzymes used in the special surface cleaning agents are especiallyproteases and amylases for removing protein- and starch-containingstains. The use of the protease triple mutant according to thisinvention in such cleaning agents for cleaning hard surfaces isrecommended especially in cases in which the susceptibility to corrosionof the contaminated material (especially in the case of light metals,such as aluminum and its alloys) makes the use of stronger acid oralkaline products impossible, for example, for cleaning membranes inhyperfiltration plants (reverse osmosis).

Conventional ingredients of this type of special cleaning agents forcleaning hard surfaces in the food industry include (in addition to theenzymes already mentioned earlier) especially components which breakdown dirt and which, as the main ingredient in such cleaning agents,serve to dissolve the dirt, mostly alkaline reacting (e.g. sodium orpotassium hydroxide, sodium or potassium carbonate) alkaline salts oforthophosphoric acid or of other organic acids, and different types ofsoda and potash water glass with different silicon dioxide/sodium oxideratios (SiO₂:Na₂O=0.7-3.3); other ingredients to be mentioned includesurface-active substances, especially for the removal of fatty dirt(anionic surfactants, e.g., fatty alcohol sulfates, alkyl benzenesulfonates and soaps; also, nonionic fatty alcohol ethers and alkylphenol glycol ethers); corrosion-protection agents, especially for useon light metals, such as aluminum and its alloys, to avoid corrosivewear, e.g., in the alkaline range, sodium silicate, and in the acidrange, a number of inhibitors that are specifically effective on acidand material; antifroth agents, e.g., paraffin oil and silicones,preferably special ethoxylation and propoxylation products.

Below, two cleaning agent compositions, in which the protease triplemutant according to this invention is used, for cleaning hard surfacesin two special fields of applications are listed:

Cleaning agent for cleaning grills:

1.0 wt % of Walocel HT 30000 PFV

0.5 wt % of Sequinon 40 Na 32

25 wt % of potassium hydroxide solution (50 wt %)

4 wt % of Rewoteric AM VSF

3 wt % of protease triple mutant (activity 300,000 DU[Delftunits]/mL±5%)

66.5 wt % of water

High-pressure active cleaning agent:

5 wt % of fatty alcohol ether sulfate (28 wt %)

10 wt % of Sequinon 40 Na 32

10 wt % of sodium hydroxide solution (40 wt %)

2 wt % of sodium cumene sulfate (40 wt %)

3 wt % of protease triple mutant (activity 300,000 DU/mL±5%)

70 wt % of water

Another application area in which the high-alkaline protease mutantsaccording to this invention can be used to advantage is the field ofleather treatment, especially the leather manufacturing industry. Inthis field of application, the protease mutant according to thisinvention is used especially in processes for hair removal skins andhides, and in particular in the main soaking cycle and/or in liming.

The protease mutants according to this invention are excellent for usein the manufacture of leather. The manufacture of leather comprises,among other things, the following steps:

a) First soaking stage for removing salt, dirt and dung

b) Main soaking stage for swelling the skin and dissolving water-solubleproteins

c) Liming for hair removal, for the principal digestion and for thesecond swelling (pelting); “pelt” is defined as the hairless digestedskin produced in this processing step

d) Deliming and tanning for removing epidermis and skin residues and forlowering the pH value

e) Pickling for acidifying the pelts

f) Chroming, i.e., mineral tannage with chromium, aluminum or zirconiumsalts

These processing steps are generally followed by a neutralization stage,possibly retannage, dyeing and greasing, and drying and dressing theleather to create ready-to-sell products. The processes mentioned above,except for drying and dressing, are carried out in the so-calledlimeyard. The containers used consist of paddles, drums, and machinesthat work according to the concrete mixer or washing machine principle.Enzymes are used in the above-mentioned processing steps up to andincluding the pickling stage. It is obvious that leather-chemicalprocesses and other applications of proteolytic enzymes in the pH rangefrom 3 to 12 require different types of enzymes to be used in theindividual processing steps since each enzyme has a pH-dependentactivity range. The range of an enzyme product for the manufacture ofleather can be considerably expanded by making the enzymes withdifferent pH activity ranges available in mixtures for use in themanufacture of leather goods. The protease mutants according to thisinvention can be used to special advantage in the leather manufacturingindustry mainly in the processing step that involves hair removal (inthe beamhouse). The mutants according to this invention arecharacterized by the fact that they ensure optimum efficacy with respectto the dissolution of the proteins responsible for hair fixation up to apH value of pH 12, especially at the low temperatures (approximately 28°C. to 30° C.) prevailing during the industrial hair removal step. Theuseful hair removal effect is additionally supported by an increased pHstability of the protease triple mutant according to this invention.Using the protease triple mutant according to this invention, the haircan be very easily removed from the skins and hides, and it is possibleto remove a very high percentage of the hair (more than approximately99%). As demonstrated in the examples of a laboratory model test, theremoval of the hair with a scraper is very easy. Thus, the proteasetriple mutants according to this invention make it possible toenzymatically remove the hair even from skins and hides of large animalscompletely and with damage in a manner that meets the practicalrequirements. Furthermore, using the protease triple mutant according tothis invention, the use of environmentally detrimental chemicals (suchas sulfide) in the beamhouse can be largely avoided.

Another subject matter of this invention quite generally concernscompositions for industrial applications which contain theabove-described high-alkaline protease according to this invention aswell as other ingredients conventionally used in such formulations for aspecific targeted application. For example, for applications such as theindustrial cleaning of hard surfaces, the compositions according to thisinvention contain the following typical ingredients: alkalis (e.g.,NaOH, KOH), anionic and nonionic surfactants, complexing agents,peroxygen bleaching agents, propylene glycol and organic solvents,phosphonates and builders. For the leather manufacturing industry,typical compositions according to this invention frequently containsodium sulfate, ammonium sulfate, sawdust and other ingredients asformulation components. These compositions are available as solidproducts which consist of mixtures of powdered constituents andgranules.

In addition to the above-described advantages of the high-alkalinebacillus protease mutants according to this invention in industrialapplications, the protease mutants according to this invention also havesurprising advantages when used in household detergent and householdercleaner compositions which contain the high-alkaline protease mutantaccording to this invention in combination with another conventionallyused prior-art protease of the “subtilisin 309”-type (which does notcontain the triple mutation N42R/N114R/N115R). The high-alkalineprotease mutant according to this invention, in a mixture with aprotease that is normally used for this particular domestic application,has a positive effect on the residual washing power after storage of thehousehold detergents. By adding the high-alkaline protease according tothis invention, the storage stability of the conventional householddetergent and household cleaner proteases is considerably increased inthe presence of the conventionally used ingredients in the detergentsand cleaning agents. In contrast to the compositions which contain theconventionally used prior-art proteases and which experience not only anobservable loss of activity but also a decrease of the washing power,the use of the protease triple mutant N42R/N114R/N115R ensures that,with respect to the conditions of universal household detergents (pH=9.5to 10.5, washing temperatures especially of 30° C. to 60° C.), anoptimum detergent effect is maintained during the storage and deliveryperiods that are normal in the distribution of these compositions, whicheffect by far exceeds the detergent effect of compositions that containonly the protease known from prior art.

This invention therefore also concerns household detergent and householdcleaner compositions which contain a high-alkaline protease mutantaccording to this invention in combination with another protease of the“subtilisin 309”-type that does not contain the triple mutationN42R/N114R/N115R. Useful household detergent and household cleanercompositions according to this invention contain the high-alkalineprotease mutant according to this invention in combination with anotherprotease, the amino acid sequence of which has a 95%, preferably a 98%,homology with respect to the amino acid sequence shown in FIG. 1, withthe other protease, however, not containing the triple mutationN42R/N114R/N115R. Especially preferred are household detergent andhousehold cleaner compositions which contain a high-alkaline proteaseaccording to this invention, the underlying amino acid sequence of whichis substantially identical to the amino acid sequence shown in FIG. 1.Preferred are those high-alkaline proteases according to this inventionwith the mutation N42R/N114R/N115R, the underlying amino acid sequenceof which is identical to the amino acid sequence shown in FIG. 1 ordiffers only in position 85 due to the natural difference asparagineinstead of serine.

The applications and compositions according to this invention shouldpreferably use those alkaline bacillus protease preparations accordingto this invention that have an enzyme activity of 50,000 to 1,000,000DU/g of enzyme preparation. “DU” is the enzymatic activity in Delftunits, where 1,000 DU correspond to the proteolytic activity which,given a volume of 1 mL of a 2% w/w enyzme solution and afterdecomposition of the casein, results in an extinction difference (1 cmlight path; 275 nm; determination against a blind test) of 4,000. Thealkaline bacillus protease preparations according to this invention canbe used in the formulations conventionally used for industrial processeseither by themselves or, optionally, in combination with other proteasesnormally used in industrial or household applications or othersubstantially conventional enzymes, such as amylases, lipases,cellulases, pectinases, nucleases, oxido reductases, etc. Relative tothe dry substance of the overall preparation, the bacillus proteasesmentioned should preferably be present in the formulations according tothis invention in quantities of 0.1 to 5 wt %, especially of 0.2 to 2 wt%.

The compositions according to this invention can be manufactured for thedifferent fields of application according to substantially known methodsin the form of a powder, e.g., in the form of granules, prills, orpellets, optionally covered with a surface coating. Due to the excellentstability of the bacillus protease mutants according to this invention,these can also be used in liquid formulations.

Under the conditions normally used in industrial processes—such ashighly alkaline pH values, e.g., pH values above 11.0, and optionallyhigh temperatures up to 70° C.—the high-alkaline bacillus proteasemutant according to this invention has surprisingly good properties.This is especially surprising since, in contrast to normal householdapplications, the application times used in industrial applications arein most cases relatively short. In addition to a high temperatureresistance, the high-alkaline bacillus protease mutant according to thisinvention has a high enzyme stability in the presence of conventionalingredients for industrial compositions as well as for householdapplications. The high-alkaline bacillus protease mutant according tothis invention is also surprisingly resistant to the bleaching agentsthat are normally used in industrial compositions or in householdapplications, such as industrial disinfecting detergents for hospitalapplications as well as household detergents.

Explanations relating to the figures:

FIG. 1:

Sequence protocol of the amino acid sequence (SEQ ID NO1) of alkalineprotease from Bacillus alcalophilus HA1 (DSM 5466).

FIG. 2:

Optimum pH value of the protease mutant in comparison with thenonmutated original protease at T=50° C.

FIG. 3:

Optimum temperature of the protease mutant in comparison with thenonmutated original protease at pH=11.

FIG. 4:

Storage stability of the protease mutant in comparison with thenonmutated starting protease in a standard detergent. The closed circlesindicate the residual activity and the solid bars indicate the detergentpower of the detergent containing the protease mutant. The open circlesindicate the residual activity and the diagonally striped bars indicatethe detergent power of the detergent containing the nonmutated startingprotease.

FIG. 5:

Detergent power of the protease mutant in comparison with the nonmutedstarting protease at pH=11.4 and T=15° C. to 60° C.

Sequencing the amino acid sequence (shown in FIG. 1) of the alkalineprotease from Bacillus alcalophilus HA1 (DSM 5466) by way of determiningthe corresponding nucleotide sequence is described in Examples 1 through4 of the European Patent Application EP 415,296. Strain HA1 of Bacillusalcalophilus was deposited on Jul. 28, 1989, with the Deutsche Sammlungvon Mikroorganismen (DSM; German equivalent of ATCC) under number DSM5466 (address: DSM—Deutsche Sammlung von Miroorganismen und ZellkulturenGmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic ofGermany).

EXAMPLES

The following examples will explain this invention in greater detailwithout in any way limiting the scope of the invention.

Example 1 Preparation of the alkaline protease which was varied bymutation in the amino acid sequence

The alkaline protease which differs from the amino acid sequence shownin FIG. 1 of the alkaline protease from Bacillus alcalophilus HA1 (DSM5466) in the amino acid exchanges N42R, N114R, and N115R was preparedaccording to substantially known methods by directed mutagenesis in DNAsubsegments of the corresponding protease gene. The numbers indicate theposition in the amino acid sequence shown in FIG. 1, with the originalamino acid preceding the numerical position in the substantially knownone-letter code and the introduced amino acid following the numericalposition. The process of directed mutagenesis for single mutations isdescribed in detail in Examples 5 through 18 of the European PatentApplication EP 415,296. Also, with respect to carrying out the processfor producing single mutations, reference is made to the examples of theEuropean Patent Application EP 503,346.

The process includes the following substantially known processing steps:

Following the method of Saito et al. (1963, Biochim. Biophys. Acta 72,pp. 619-629), chromosomal DNA was isolated from the natural isolateBacillus alcalophilus HA1 (DSM 5466) and partially hydrolyzed with therestriction endonuclease Sau3A. The restriction fragments werefractionated by means of electrophoresis and the fragments were isolatedin sizes of 3 to 8 kilobases. The isolated and size-selected DNAfragments from Bacillus alcalophilus HA1 were newly combined in vitrowith vector DNA of the substantially known plasmid pUB 110 according tosubstantially known methods. Using the method described by S. Chang andN. Cohen (1979, Mol. Gen. Genet. 168, pp. 111-115), protoplasts of thestrain Bacillus subtilis BD224 (Bacillus Genetic Stock Center 1A46) weretransformed with the obtained DNA that was newly combined in vitro. Thetransformants were selected on plates with neomycin. According to themanual by Maniatis et al. (Maniatis et al.=T. Maniatis, E. F. Fritsch,J. Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, 1982), the plasmid DNA was isolated from a clone. Thefragment from B. alcalophilus DNA that was contained in this plasmid hada size of 4.1 kilobases and contained the complete DNA sequence for thehigh-alkaline protease from Bacillus alcalophilus HA1 (DSM 5466) (seeExamples 1 and 2 of the European Patent Application EP 415,296).

The plasmid which contained the complete DNA sequence for thehigh-alkaline protease from Bacillus alcalophilus HA1 (DSM 5466) was cutwith AvaI. The protruding ends were filled according to substantiallywell-known methods (Maniatis et al., p. 114) to form a DNA doublestrand. After subsequently restricting this DNA with XbaI, theN-terminal fragment comprising 1.618 BP was isolated and clonedaccording to substantially known methods into the vector pBS. Theresulting vector contained the N-terminal end for the DNA that codes forthe amino acid sequence shown in FIG. 1 (see Example 5 of the EuropeanPatent Application 415,296).

Using a similar method, a vector was created which contained a DNAfragment which comprises 658 BP and which codes for the C-terminal endof the corresponding protease. For this purpose, the plasmid thatcontained the complete DNA sequence was cut with the restrictionendonucleases XbaI and Asp718 and cloned into the cutting site of theknown vector pBS (see Example 7 of the European Patent Application415,296).

Using the “primer extension” technique described by T. A. Kunkel (1985,Proc. Natl. Acad. Sci. USA 82, pp. 488-492), the directed mutations werecarried out in the DNA subsequence that contained the C-terminal or theN-terminal end. For this purpose, the corresponding vectors were firsttransformed according to substantially known methods into theiruracilated single-strand analogs by cultivating transformed E. coliCJ236 bacteria which were additionally infected with the helper phageM13 K07 (obtained from Bio-Rad Laboratories, Richmond, Calif.), with oneof the two vectors. The bacterium E. coli CJ236 is [word or wordsmissing] to a substantially known defective uracil-N-glycosylase whichduring the replication of the vectors, incorporates the nucleotideuracil instead of thymidine into the DNA sequence of the vector.Uracilated vectors can be used to advantage according to substantiallyknown methods for in-vitro reactions of the directed mutagenesis sincethe uracil-containing single DNA strand which served as a matrix togenerate mutated DNA strands can be removed after conclusion of thereactions by treating it with uracil-N-glycosylase. The use of thehelper phages mentioned was required for the synthesis of the envelopeproteins for the uracilated single-strand vector DNA that formed.Uracilated single-strand envelope vector DNA was extracted from thetransformed host organism E. coli CJ236 and subsequently isolated fromthe culture medium.

The isolated uracilated DNA single-strand vectors of the C-terminal orN-terminal end were hybridized with synthetic oligonucleotides whichcontained the mutation sites and, at the same time, served as primersfor the subsequent completion of the complete DNA double strand withmutation. The synthetic oligonucleotides used were produced according tosubstantially known methods according to the process described by S. L.Beaucage and M. H. Caruthers (1981, Tetrahedron Letters 22, pp.1859-1862). The synthesis of the second DNA strand was carried outaccording to substantially known methods by means of T4 DNA polymeraseand subsequent ligation with T4 DNA ligase (Kunkel et al., 1987, Methodsin Enzymol. 154, pp. 367-382). The double-strand vector DNA that formedwas transformed in E. coli MC 1061 and the mutated vectors wereidentified by examining the corresponding unitary restrictionendonuclease recognition sites which were introduced or removed with thesynthetic oligonucleotides.

To produce, e.g., two mutations either in the N-terminal or in theC-terminal portion of the protease DNA, the process, after introducing afirst mutation, was repeated by using a further syntheticoligonucleotide to introduce a second mutation.

Expression vectors with mutations in the C-terminal portion or theN-terminal portion of the protease DNA sequence were produced by cuttingthe DNA sequences, which were obtained by means of the directedmutagenesis, with restriction endonucleases and by ligating them with avector DNA which contained the other terminal portion of the DNAsequence and all elements required for the expression. The vectorsobtained were complete expression vectors with a suitable reading framefor the expression of the correspondingly mutated protease. Expressionsvectors of this type were produced following the method that isexplained in greater detail in Example 16 of the European PatentApplication EP 415,296. For the subsequent triple mutation, theexpression vector DSM 5466 Mut. N42R/N114R/115R was produced using thesame process.

The triple-mutated high-alkaline protease was produced by transformingand cultivating B. subtilis BD 224 with the above-mentioned expressionvector using a substantially known method. The triple-mutatedhigh-alkaline protease was isolated from the protruding culture of thetransformed and cultivated strain according to well-known methods. Adetailed description of the method for isolating mutated proteases canbe found in Examples 16 and 18 of the European Patent Application EP415,296 on which this work was based.

The alkaline protease on the basis of Bacillus alcalophilus HA1(DSM5466) which was varied as a result of the triple mutation in theamino acid sequence was used in the tests described in Examples 2through 4.

The application and the advantages of the triple mutant according tothis invention in industrial textile laundering processes are describedin greater detail in the examples of the parallel andpriority-establishing German Patent Application DE 4,411,223 to whichreference is hereby made.

Other properties of the protease mutant according to this invention arelisted in Example 5.

Example 2 Determination of the hair removal property of the proteasetriple mutant according to this invention

Preliminary cleaning:

The cowhide is soaked in twice the quantity of water (relative to thecowhide) for 1 h with 0.1% of Marlipal 013/939 at approximately 28°C.-30° C. The cowhide is subsequently cut into pieces measuringapproximately 20×60 cm and stored at −20° C. From these stored cowhidepieces, test samples measuring approximately 20×7 cm are cut, the excesswater is allowed to drip off, and the samples are dried with papertowels. The test samples are [word or words missing; probably:incubated] for 120 min in a solution with 0.5% soda and 0.1% [word orwords missing] stirred for 15 min. The increase in weight is recorded.

A. Swelling (water absorption) of the precleaned hides Incubationsolution: 0.5% Na carbonate, 0.1% Marlipal (relative to the hide), twicethe quantity of water (relative to the hide) Incubation time: 120 minIncubation temperature: 28° C. Enzyme: Optimase L660 Weight WeightDifference Enzyme Dosage after before increase in (g/100 kg incubationincubation weight Test cowhide) [AADU/kg] [g] [g] [g] [%] 1 6,08 37470729,53 701,59 27,940 4,0 B. Hair removal Incubation solution: 7.1 g/LCa(OH)2 (200% relative to the cowhide) Incubation time: 24 h Incubationtemperature: 30° C. Mixing: At the beginning of the incubation, 2 hcontinuously in the Linitest machine and at the end, 3.5 hourscontinuously pH value: Beginning: 12.48 End: 12.45 Container Removal ofNo. Protease Enzyme Dosage the Skin 1 Blind Test — — 3 2 Blind Test — —3 3 Mutant 310,81 925188 1 4 Mutant 310,81 925188 1 Mutant = proteasetriple mutant N42R/N114R/N115R with activity of 300.000 DU/mL (+5%) 1:Easy and complete removal of the hair 2: Almost complete removal of thehair but still some (lumpy) residues on the skin 3: Only very few hairsor no hairs at all are removed.

Commentary: After processing step A, the test samples were soft andspongy, after processing step B, the test samples had “hardened into arubberlike texture.”

Example 3 Origin of the test samples: cow, back

Preliminary cleaning:

The cowhide is soaked in twice the quantity of water (relative to thecowhide) for 1 h with 0.1% of Marlipal 013/939 at approximately 28°C.-30° C. The cowhide is subsequently cut into pieces measuringapproximately 20×60 cm and stored at −20° C. From these stored cowhidepieces, test samples measuring approximately 20×7 cm are cut, the excesswater is allowed to drip off, and the samples are dried with papertowels. The test samples are [word or words missing; probably:incubated] for 120 min in a solution with 0.5% soda and 0.1% [word orwords missing] stirred for 15 min. The increase in weight is recorded.

Under the conditions listed below, 3 tests were carried out.

A. swelling (water absorption) of the precleaned hides Incubationsolution: 0.5% Na carbonate, 0.1% Marlipal (relative to the hide), twicethe quantity of water (relative to the hide) Incubation time: 120 minIncubation temperature: 28° C. Enzyme: Optimase L660 Weight WeightDifference Enzyme Dosage after before increase in (g/100 kg incubationincubation weight Test cowhide) [AADU/kg] [g] [g] [g] [%] 1 15,89 20060715,98 732,58 −16,60 −2,3 2  3,26 20122 905,27 866,22  39,05  4,5 3 3,32 20476 338,39 342,25  −3,86 −1,1 ** Between step A and B, the testspecimens were stored in the refrigerator for 1.5 h. B. Hair removalIncubation solution: 7.1 g/L Ca(OH)₂ (200% relative to the cowhide)Incubation time: 24 h Incubation temperature: 30° C. Mixing: At thebeginning of the incubation, 2 h continuously in the Linitest machineand at the end, 3.5 hours continuously pH value: Beginning: End: Test 112.48 12.46 Test 2 12.55 12.53 Test 3 12.5 12.5 Results of test 1:Protease used: Mutant N42R/N114R/N115R with an activity of 300.000 DU/mL(±5%) Container Enzyme Dosage Removal of No. (g/100 kg cowhide) [AADU/kgcowhide] the Skin* 1 — — 3   2 — — 3   3 31,2 92745 1,4 4 31,2 92745 1,2

Commentary: A certain amount of the hair was uniformly removed but astubble of approximately 1-3 mm remained.

Results of test 2 Protease used: Mutant N42R/N114R/N115R with anactivity of 300.000 DU/mL (±5%) Container Enzyme Dosage Removal of No.(g/100 kg cowhide) [AADU/kg cowhide] the Skin* 1  10,1  29969 2,2  2 50,3 149844 1,8  3 100,7 299688 2,0** 4 151,0 449531 1,4** ** Neckportion with a stronger growth of hair

Results of test 3 Protease used: Mutant N42R/N114R/N115R with anactivity of 300,000 DU/mL (±5%) Container Enzyme Dosage Removal of No.(g/100 kg cowhide) [AADU/kg cowhide] the Skin* 1 151,3 450294 1,2 2100,9 300196 1,4 3  50,4 150098 1,6 Legend referring to Test 1 to 3 1:Easy and complete removal of the hair 2: Almost complete removal of thehair but still some (lumpy) residues on the skin 3: Only very few hairsor no hairs at all are removed.

Example 4 A. Main soaking cycle

Cowhide (weight): 1886.25 g (precleaning same as in Example 2 and 3)Body part: Belly Formulation: Water: 3772.50 g Na₂CO₃: 3.43 gSurfactant: 1.89 g Marlipal Enzyme: 0.4046 g optimase L660 correspondsto: 99582 AADU/kg cowhide Weight after a reaction for 4 h: 2039.5 gIncrease in weight: 8.1 wt % pH value after 0 reaction for 4 h: 10.21 B.Hair removal Water: 3059.25 g Ca(OH)2 20.40 g Enzyme: 6.9352 g proteasemutant with an activity of 150,000 DU/mL (±5%) corresponds to: 539878AADU/kg cowhide

pH value after an incubation over 17.25 h: 12.75

Visual inspection of the hair removal results:

It was very easy to remove the hair; when the test sample was removedfrom the drum, the hair fell out as soon as the opening edge wastouched. Only 2-3 areas measuring approximately 2 cm² remained but itwas easy to remove the hair with a scraper.

Example 5

Below, a few other important properties of the triple mutant accordingto this invention will be explained.

a) The activity of the triple mutant was determined in comparison to thenonmutated original protease as a function of the pH value; thefollowing conditions prevailed: substrate=acetyl casein, T=50° C.,reaction time=10 min, phosphate borate buffer. The pH-dependent relativeactivity in % (the activity at pH=8.5 is defined as 100%) is shown inFIG. 2. At higher pH values in a range from pH=10 to 12, the triplemutant according to this invention is considerably more active than theoriginal protease.

b) The activity of the triple mutant according to this invention incomparison to the nonmutated original protease as a function of thetemperature was determined under following conditions: substrate=acetylcasein, pH=11. The temperature-dependent relative activity in % (theactivity at pH=8.5 and T=50° C. is defined as 100%) is shown in FIG. 3.At the high pH value chosen and in the temperature range from 45° C. to58° C., the triple mutant according to this invention is considerablymore active than the original protease.

c) The shelf life of the triple mutant according to this invention incomparison with the nonmutated original protease as a function of theshelf life was determined by storing the triple mutant in a standardheavy-duty detergent under the following conditions: T=30° C., 60%relative humidity (r.h.), in closed boxes. The residual activity in %determined after storage (relative to the original activity=100%) of thetriple mutant according to this invention is considerably higher, thusalso improving the stability. The results are shown in FIG. 4.

d) To demonstrate the high-quality cleaning effect (cleaning hardsurfaces) of the triple mutant according to this invention, thedetergent power was compared to that of the original protease anddetermined for different enzyme concentration under the followingconditions: standard detergent base; pH=11.4 (6 g/L); test fabric=EMPA117, T=60° C. The results found (delta reflectance) as a function of theenzyme quantity used (mg/L) are illustrated in FIG. 5, which shows thesuperiority of the protease according to this invention at high pHvalues and an increased temperature.

e) To demonstrate the advantages of the combination of the triple mutantaccording to this invention with the prior-art detergent proteases (inthis case, the original protease of the “subtilisin 309”-type), thefollowing tests were carried out which led to the results indicated.

Test 1

Relative detergent power of the triple mutant according to thisinvention in comparison with the nonmutated original protease (coveredwith a coating) prior to and after storage in a heavy-duty detergent forhousehold applications. This detergent power is the average detergentpower obtained from 2 test fabrics with doses of the same activity.

After 6 weeks of storage Protease Prior to storage at 35° C./80% r.h.Original protease 97,15 28,15 Triple mutant 66,22 50,72 N42R/N114R/N115R61,60 50,79

Test 2

Residual activity and residual detergent power after storage of thetriple mutant according to this invention an the original protease indetergent powder at 30° C. and 60% r.h. (relative humidity); pH valueprior to washing is 10.2.

Storage time A B C D 0 Weeks 100,0 24,50 100,0 22,99 1 Week   92,4 —100,0 — 2 Weeks  86,9 —  94,6 — 4 Weeks  75,1 —  86,0 — 6 Weeks  60,914,98  70,8 21,63

Test fabric: Blood/milk/ink (EMPA 117)

% dR: Difference between the reflection of the test fabric(enzyme-containing detergent) and the reflection of the test fabric thatwas washed with enzyme-free detergent base.

Enzyme dose: Same activity

A=Residual activity (%), original protease

B=Detergent power (% dR), original protease

C=Residual activity (%), triple mutant

D=Detergent power (% dR), triple mutant

Definitions

Optimase L660=alkaline protease from B. licheniformis

Marlipal=polyethylene glycol isotridecyl ether

Walocel=2-hydroxyethyl cellulose ether

Sequinon=[[bis[2-[bis(phosphonomethyl)amino]ethyl]amino]-methyl]phosphonic acid, sodium salt

Rewoteric=ampholyte on the basis of imidazoline

EMPA117=test fabric of polyester/cotton with contamination ofblood/milk/ink (Swiss Materials Testing Institute)

1 380 amino acids amino acid single linear unknown 1 Met Lys Lys Pro LeuGly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile -110 -105 -100 Ser Val AlaPhe Ser Ser Ser Ile Ala Ser Ala Ala Glu Glu Ala Lys -95 -90 -85 -80 GluLys Tyr Leu Ile Gly Phe Asn Glu Gln Glu Ala Val Ser Glu Phe -75 -70 -65Val Glu Gln Val Glu Ala Asn Asp Glu Val Ala Ile Leu Ser Glu Glu -60 -55-50 Glu Glu Val Glu Ile Glu Leu Leu His Glu Phe Glu Thr Ile Pro Val -45-40 -35 Leu Ser Val Glu Leu Ser Pro Glu Asp Val Asp Ala Leu Glu Leu Asp-30 -25 -20 Pro Ala Ile Ser Tyr Ile Glu Glu Asp Ala Glu Val Thr Thr MetAla -15 -10 -5 1 Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro AlaAla His 5 10 15 Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val LeuAsp Thr 20 25 30 Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly AlaSer Phe 35 40 45 Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His GlyThr His 50 55 60 65 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile GlyVal Leu Gly 70 75 80 Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val LeuGly Ala Ser 85 90 95 Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu GluTrp Ala Gly 100 105 110 Asn Asn Gly Met His Val Ala Asn Leu Ser Leu GlySer Pro Ser Pro 115 120 125 Ser Ala Thr Leu Glu Gln Ala Val Asn Ser AlaThr Ser Arg Gly Val 130 135 140 145 Leu Val Val Ala Ala Ser Gly Asn SerGly Ala Gly Ser Ile Ser Tyr 150 155 160 Pro Ala Arg Tyr Ala Asn Ala MetAla Val Gly Ala Thr Asp Gln Asn 165 170 175 Asn Asn Arg Ala Ser Phe SerGln Tyr Gly Ala Gly Leu Asp Ile Val 180 185 190 Ala Pro Gly Val Asn ValGln Ser Thr Tyr Pro Gly Ser Thr Tyr Ala 195 200 205 Ser Leu Asn Gly ThrSer Met Ala Thr Pro His Val Ala Gly Ala Ala 210 215 220 225 Ala Leu ValLys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg 230 235 240 Asn HisLeu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr 245 250 255 GlySer Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265

What is claimed is:
 1. An industrial process comprising cleaning a hardsurface using a modified subtilisin protease wherein a substitution ofarginine for the amino acids occurs at each of the amino acid positionshomologous to positions 42, 114 and 115 of SEQ ID NO:
 1. 2. The processof claim 1, wherein said hard surface is in an industrial plant.
 3. Theprocess of claim 1, wherein said hard surface is in a location in thefood industry.
 4. The process of claim 3, wherein said location selectedfrom the group consisting of slaughtering plants, institutionalkitchens, and grill restaurants.
 5. A cleaning process comprising theapplication of a modified subtilisin protease to the manufacture ofleather, wherein said modified subtilisin protease comprises asubstitution of arginine for the amino acids occurring at each of theamino acid positions homologous to positions 42, 114 and 115 of SEQ IDNO:
 1. 6. The process of claim 5, wherein said application of saidmodified subtilisin protease is used for removing hair from skin orhide.
 7. A composition for industrial applications comprising a modifiedsubtilisin protease and at least one other ingredient conventionallyused in formulations for cleaning hard surfaces, wherein said modifiedsubtilisin protease comprises a substitution of arginine for the aminoacids occurring at each of the amino acid positions homologous topositions 42, 114 and 115 of SEQ ID NO:
 1. 8. The composition of claim7, wherein said at least one other ingredient comprises an ingredientselected from the group consisting of a component which dissolves dirt,a compound for the removal of fatty acids, a corrosion-protection agentand an antifroth agent.
 9. The composition of claim 8, wherein saidcomponent which dissolves dirt is selected from the group consisting ofsodium hydroxide, potassium hydroxide, sodium carbonate, potassiumcarbonate, an alkaline salt of orthophosphoric acid, and soda and potashglass.
 10. The composition of claim 8, wherein said compound for theremoval of fatty acids is an alkyl phenol glycol ether.
 11. Acomposition for use in the manufacture of leather comprising a modifiedsubtilisin protease and at least one other ingredient conventionallyused in formulations for manufacturing leather, wherein said modifiedsubtilisin protease comprises a substitution of arginine for the aminoacids occurring at each of the amino acid positions homologous topositions 42, 114 and 115 of SEQ ID NO:
 1. 12. The composition of claim11, wherein said at least one other ingredient comprises an ingredientselected from the group consisting of sodium sulfate, ammonium sulfate,calcium hydroxide, sodium carbonate, soda, a surfactant, and sawdust.